Reagent and method for the quantitative determination of bilirubin



United States Patent 3,348,920 REAGENT AND METHOD FOR THE QUANTITA- TIVE DETERMINATION OF BILIRUBIN Patrick V. Ferro, Miami, and Anna Bell Ham, Coral Gables, Fla, assignors to Dade Reagents, Inc., Miami, Fla, a corporation of Florida N0 Drawing. Filed Feb. 25, 1964, Ser. No. 347,121 17 Claims. (Cl. 23-230) ABSTRACT OF THE DISTILOSURE A reagent and method for the quantitative determination of total serum bilirubin. A single stable reagent containing a ferric ion catalyst, a water-miscible alcohol, an acid capable of oxidizing bilirubin in the presence of ferric ions, and a water-miscible lipid solvent comprising an ethylene glycol derivative having one of the hydroxyl groups thereof etherified by a short chain alkyl group, is added to the serum. Following precipitation of the serum protein, the blue-colored filtrate may be read spectrophotometrically to determine total serum bilirubin quantitatively.

This invention relates to the quantitative determination of the bile pigment bilirubin in aqueous and organic fluids, and particularly to the determination of total bilirubin in human or animal serum. Such determination of serum bilirubin is useful in measuring the extent and progress of diseases of blood, liver, and other organs in which jaundice may be present.

While it has long been known that the presence of bilirubin in solution may be qualitatively detected by acid oxidation, such a procedure has never been successfully used for the quantitative estimation of total bilirubin in serum. The principal barriers to such a quantitative determination of serum bilirubin through oxidation have been the difficulties encountered in removing all of the bilirubin from the precipitate protein so that the bilirubin would remain in the liquid layer above the precipitate, the occurrences of side reactions which produce turbidity in the final solution, and the non-linearity of the reactions resulting from too greatly accelerated or uncontrolled oxidation.

In the absence of a more satisfactory procedure for the quantitative estimation of serum bilirubin, workers have for the past fifty years or more resorted to a method based on the diazo reaction employing sulfanilic acid, sodium nitrite, and hydrochloric acid. The principle of such a test is the coupling of bilirubin with diazotized sulfanilic acid (sulfobenzenediazonium). The result of this coupling is a compound, purple in color, known as ambilirubin. The intensity of the purple color is then measured in a colorimeter or spectrophotometer to estimate the bilirubin concentration.

Among the shortcomings of the diazo reaction for the determination of bilirubin are the instability of the final color and the inaccuracies that might result from such instability, the need for several reagents which must be mixed shortly before the test, the time required in preparing for and in conducting such tests, and the inaccuracies which result when compounds in the serum other than bilirubin respond to diazotization.

Therefore, it is a principal object of the present invention to provide a reagent and method for overcoming the aforementioned defects and disadvantages of prior reagents and procedures. Specifically, it is an object to provide a single stable reagent which may be added to serum and which will oxidize all of the bilirubin thereof to produce a colored solution suitable for quantitative colorimetric analysis.

A further object of the present invention is to provide a reagent and bilirubin testing method which not only oxdizes the bilirubin but which also extracts the bilirubin from the protein of the serum, separates the serum globulin from the albumin, and solubilizes the lipids extracted from the serum that would, in the absence of such solubilizing, cause turbidity and so prevent quantitative colorimetric analysis. Other objects will appear as the specification proceeds.

The reagent embodying the present invention contains four constituents; namely, an acid capable of oxidizing bilirubin to biliverdin in the presence of ferric ions, a primary catalyst as a source of ferric ions, a water-miscible alcohol, and a lipid solubilizing agent miscible with water and with the other constituents.

The acid component may be any organic or inorganic acid which, in the presence of ferric ions, is capable of oxidizing bilirubin such as, for example, hydrochloric acid or glacial acetic acid. Trichloroacetic acid has been found particularly suitable although other acids having similar bilirubin-oxidizing properties (in the presence of a ferric ion catalyst) may be used. If desired, two or more of such acids may be combined. Thus, the acid constituent of the reagent may comprise a combination of trichloroacetic acid and hydrochloric acid.

Bilirubin, when reacted with acid in the presence of ferric ions, oxidizes to biliverdin and/ or cholecyanin, the reaction product being blue or blue-green in color and the intensity of the color being related to the extent of reaction which is in turn dependent upon the amount of bilirubin originally present. Any material which contains or liberates ferric ions may be used as the catalyst source, ferric chloride being particularly suitable for this purpose.

An important aspect of the invention lies in providing a reagent and method which bring about a complete release of all of the serum bilirubin from the serum protein prior to, or at the same time that, the protein is being precipitated. It is believed that approximately 25 percent of the bilirubin is released from the protein by the addition of the acid, the remaining percent being extracted by the addition of the alcohol. Isopropyl alcohol is particularly effective; however, as set forth in the accompanying examples, other water-miscible alcohols such as ethyl alcohol or methyl alcohol, or even an alcohol having lower water miscibility such as butyl alcohol, may also be used.

The alcohol, acting as the solvent of the system, also extracts from the serum the fatty components and with the water that is present from the serum causes the extract to be turbid. To prevent such turbidity, We employ as a lipid solvent an ethylene glycol derivative in which one of the hydroxy groups is etherified by a short chain alkyl group. Examples of such ethylene glycol derivatives which may be used effectively in practicing the invention are ethylene glycol monomethyl ether, ethylene glycol monoethyl ether, ethylene glycol monobutyl ether, diethylene glycol monomethyl ether, diethylene glycol monoethyl ether, and diethylene glycol monobutyl ether. All of such derivatives are characterized by the fact that they are water miscible, are ethers of ethylene or diethylene glycol, and contain alkyl groups having between 1 to 4 carbon atoms.

While the ferric ion is the primary catalytic agent for bilirubin oxidation, the ether derivative of ethylene glycol also performs a significant function as a secondary catalyst to accelerate the reaction to completion. Ferric iron concentrations of 10 to 5,000 micrograms per milliliters of serum have been found effective in catalyzing the reaction; however, for proper control of the rate at which oxidation takes place and for insuring the production of a stable blue clr, a ferric iron concentration of between 500 to 2,000 micrograms per 100 milliliters of serum is pre-v ferred. The optimum ferric iron concentration has been found to be approximately 1500 micrograms per 100 milliliters of test solution. When using the latter concen tration, the ferric ion will catalyze the reaction to comletion within about to 30 minutes and the final bluecolored solution will be stable for approximately two hours. Increased amounts of ferric ion, about 5,000 micrograms of iron per 100 milliliters of serum, will accelerate the reaction with an undiluted serum containing 20 milligrams of bilirubin per 100 milliliters solution to compleshould be approximately 5 to 15 percent. There may be a considerable variation within these ranges, although the preferred concentration range for the acid constituent is approximately 5 to percent by Weight; The preferred ratio of reagent to serum is 10 to 1; this ratio appears to give optimum results for deproteinization, extraction of bilirubin, clarity of the final solution, and rate of oxidation of bilirubin. However, other ratios of 1 volume of serum and up to 20 volumes of reagent may be used.

The reagent, prepared as set forth in the accompanying illustrative examples and composed of the four constituents already described, is used as follows: 1 part of serum (or an appropriate aqueous bilirubin solution) is pipettcd into a test tube, followed by 10 parts of the bilirubin testing reagent. The tube-should be inverted several times to mix the contents and should then beallowed to stand for 3 to 5 minutes topermit precipitation of the serum protein. Thereafter, the contents of the tube are filtered or centrifuged, and the filtrate is transferred to a cuvet. To insure full color development, the filtrate is allowed to stand for approximately 30 minutes from the time of addition of the reagent. The color, optical density, or percentage of light transmittance is thereafter determined using a suitable instrument such as a colorimeter or spectrophotometer.

The chromogenic reaction of bilirubin with the reagent of the present invention proceeds more uniformly, and the final blue-colored solution is much more stable, than that of the diazo type reaction customarily used for bilirubin determinations, resulting in considerably greater accuracy and reproducibility of such determinations.

A-more complete understanding of the reagent and method of the invention may be obtained by reference to the following illustrative examples:

Another reagent embodying the invention may be pre-.

pared by placing 100 grams of trichloroacetic acid in a 100 milliliter graduated mixing cylinder, then dissolving the trichloroacetic acid in about 500 milliliters of ethyl alcohol. To this mixture is then added 1 milliliter of a ferric chloride stock solution. [which contains 7.2 grams 4 of ferric chloride (FeCl 61-1 0) in 100 milliliters of water and which contains 1,500 micrograms of ferric iron per 1 milliliter]. To this solution is then added 100 milliliters of ethylene glycol monoethyl ether. Thereafter, the volume is adjusted to 1,000 milliliters with. ethyl alcohol and thoroughly mixed.

III

Diagnostic reagents suitable for reaction with, serum bilirubin in the manner recited in the foregoing specification, are prepared according to the procedure and proportions given in Example I with the following constituents substituted for the ingredients specified in such example:

Acid Alcohol Lipid Solvent 1 Hydrochlorim- Isopropyl. Ethylene glycol monomethyl ether. 2 do. Butyl Ethylene glycol monobutyl ether. 3 do.... Methyl Diithylene glycol monornethyl e or. Glacial acetic Isopropyl. Diethylcne glycol monoethyl ether.

Ethyl Ethylene glycol monoethyl ether. 0 Butyl Diethylene glycol mouobutyl ether 1. A diagnostic reagent for the quantitative determina tion of bilirubin comprising an acid capable of oxidizing bilirubin in the presence of ferric ions, a ferric ion catalyst, a water-miscible alcohol, and a water-miscible lipid solvent comprising an ethylene glycol derivative having one of the hydroxyl groups thereof etherified by a shortchain alkyl group.

2. The reagent of claim 1 in which said ethylene glycol derivative is ethylene glycol monoalkyl ether.

3. The reagent of claim 1 in whichsaid ethylene glycol derivative is diethylene glycol monoalkyl ether.

4. Y The diagnostic reagent of claim 1 in which said ferric ion catalyst comprises ferric chloride in the proportion within the range of 10 to 5,000 micrograms of ferric iron for each 100 milliliters of reagent.

5. The reagent of claim 4 in which the proportion of ferric chloride is within the preferred-range of 500 to 2,000 micrograms of ferric iron for each. 100 milliliters of reagent.

6. A diagnostic reagent forthe quantitative estimation of total serum bilirubin comprising 2 to 20 percent of an acidcapable of oxidizing bilirubin in the presence of ferric ions, 70 to percent of a water-miscible alcohol, 5 to 15 percent of a water-miscible lipid solvent comprising an ethylene glycol derivative having one of the hydroxylgroups thereof etherified by a short-chain alkylgroup, and

a ferric ion catalyst containing ferric iron within the range of 500 to 2,000 micrograms per milliliters of reagent.'

7. The reagent of claim 6 in which said alcohol is isopropyl alcohol.

8. The reagent of claim 6 in which said alcohol is ethyl alcohol.

9. The reagent of claim 6 in which said alcohol is methyl alcohol.

10. The reagent of claim 6 in which said alcohol is butyl alcohol.

capable of oxidizing bilirubin in the presence of ferric ions, a ferric ion catalyst, an alcohol, and a lipid solvent comprising an ethylene glycol derivative having one of the hydroxyl groups thereof etherified by a short chain alkyl group, to solubilize and oxidize all of said bilirubin; and thereafter colorimetrically analyzing the oxidized bilirubin solution to determine optically the bilirubin content thereof.

15. The method of claim 14 in Which said sample and reagent are reacted in the proportion of approximately 1 to 10 by volume.

16. A method of quantitatively determining the bilirubin content of serum comprising the steps of mixing one volume of serum with an excess of up to approximately 20 volumes of a diagnostic reagent containing a watermiscible alcohol, an acid capable of oxidizing bilirubin in the presence of ferric ions, a Water-miscible lipid solvent comprising an ethylene glycol derivative having one of the hydroxyl groups thereof etherified by a short chain alkyl group, and a ferric ion catalyst; maintaining the reactants in contact until serum protein is precipitated and serum bilirubin is oxidized and in solution, and thereafter separating said solution from said precipitate.

6 17. The method of claim 16 in which there is the further step of analyzing said solution colorimetrically to determine quantitatively the bilirubin content thereof.

References Cited Ferme: Detection and Rapid Determination of Bilirubin, Chemical Abstracts, vol. 55, p. 9548a, May 1961.

Fog. et al.: Determination of Bilirubin and Other Tetrapyrroles, Chemical Abstracts, vol. 57, p. 11475H, October 1962.

Mathieu-Daude et al.: Spectrophotometry of Bilirubin (PAC. Med. Montpellier, France), Montpellier Med. 61(2), 155260 (1962), reported in Chem. Abs., vol. 60, p. 12343, May 1964.

Stevenson et al.: spectrometric Determination of Free and Total Bilirubin in Serum, Clin. Chem. 10, 95- 102 (Feb. 4, 1964), (Reported in Chemical Abstract, vol. 60, p. 13556, May 1964).

MORRIS O. WOLK, Primary Examiner. L. MEI, Assistant Examiner. 

14. A METHOD FOR QUANTITATIVELY DETERMINING THE BILIRUBIN CONTENT OF A SAMPLE COMPRISING THE STEPS OF REACTING SAID SAMPLE WITH A DIAGNOSTIC REAGENT CONTAINING AN ACID CAPABLE OF OXIDIZING BILIRUBIN IN THE PRESENCE OF FERRIC IONS, A FERRIC ION CATALYST, AN ALCOHOL, AND A LIPID SOLVENT COMPRISING AN ETHYLENE GLYCOL DERIVATIVE HAVING ONE OF THE HYDROXYL GROUPS THREREOF ETHERIFIED BY A SHORT CHAIN ALKYL GROUP, TO SOLUBILIZE AND OXIDIZE ALL OF SAID BILIRUBIN; AND THEREAFTER COLORIMETRICALLY ANALYZING THE OXIDIZED BILIRUBIN SOLUTION TO DETERMINE OPTICALLY THE BILIRUBIN CONTENT THEREOF. 